贾凡, 毛自朝, 王志远, 赵静, 吴毅歆, 何月秋. 枯草芽孢杆菌XF1菌株中XFsacA基因的克隆及功能验证[J]. 华南农业大学学报, 2012, 33(2): 154-158. DOI: 10.7671/j.issn.1001-411X.2012.02.007
    引用本文: 贾凡, 毛自朝, 王志远, 赵静, 吴毅歆, 何月秋. 枯草芽孢杆菌XF1菌株中XFsacA基因的克隆及功能验证[J]. 华南农业大学学报, 2012, 33(2): 154-158. DOI: 10.7671/j.issn.1001-411X.2012.02.007
    JIA Fan, MAO Zi-chao, WANG Zhi-yuan, ZHAO Jing, WU Yi-xin, HE Yue-qiu. Cloning and Functional Confirmation of XFsacA Gene from Bacillus subtilis XF1[J]. Journal of South China Agricultural University, 2012, 33(2): 154-158. DOI: 10.7671/j.issn.1001-411X.2012.02.007
    Citation: JIA Fan, MAO Zi-chao, WANG Zhi-yuan, ZHAO Jing, WU Yi-xin, HE Yue-qiu. Cloning and Functional Confirmation of XFsacA Gene from Bacillus subtilis XF1[J]. Journal of South China Agricultural University, 2012, 33(2): 154-158. DOI: 10.7671/j.issn.1001-411X.2012.02.007

    枯草芽孢杆菌XF1菌株中XFsacA基因的克隆及功能验证

    Cloning and Functional Confirmation of XFsacA Gene from Bacillus subtilis XF1

    • 摘要: 枯草芽孢杆菌Bacillus subtilis XF1是从土壤中分离的一株利用蔗糖快、能高效防治根肿病的专利菌株.为探明其高效的蔗糖代谢机制,用PCR从该菌中扩增到蔗糖代谢关键基因蔗糖-6-磷酸水解酶基因(XFsacA基因),其编码的蛋白质氨基酸序列与枯草芽孢杆菌B168菌株中的XFsacA基因有97%的相似性,且发生了12个氨基酸突变.将该基因中约1.4 kb的编码框序列连接到表达载体pQE30上,构建了重组表达质粒pQE30-XFsacA,该质粒转入到不能利用蔗糖的Escherichia coli BL21中,后者能在以蔗糖为唯一碳源的M9无机离子培养基中正常生长,并表达出了一条约54 000蛋白条带.结果预示XFsacA基因氨基酸序列的替代可能是XF1高效利用蔗糖的基础,同时XFsacA基因的克隆与其在E. coli中的表达研究,为利用蔗糖发酵的工程E. coli构建奠定了基础.

       

      Abstract: Bacillus subtilis XF1, which is patented and a very efficient biocontrol agent to control clubroot disease of cruciferous crops, grows very quickly when sucrose is used as carbon source. To elucidate the growth promotion mechanism of sucrose, the XFsacA gene, encoding a key enzyme for sucrose metabolism, was amplified by PCR and sequenced. Its amino acid sequence has 97% homolog to Bacillus subtilis B168 except 12 amino acids replacement. The coding region of the gene was inserted into plasmid pQE30 to construct an expression plasmid, pQE30-XFsacA. After pQE30-XFsacA was transformed into Escherichia coli BL21, which could not use sucrose, a protein band about 54 000 detected by SDS PAGE analysis indicated the gene was successfully expressed in BL21. The function of XFsacA gene was confirmed by the transformed BL21 having the ability to grow and survive in M9 medium with sucrose as sole carbon source. The data indicated that the replacement of 12 amino acids of XFsacA in XF1 strain might improve the efficiency in use of sucrose,and cloning and functional expression of XFsacA in E. coli could be a good strategy for developing metabolic engineered E. coli strain to use sucrose.

       

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